Programmed −1 ribosomal frameshifting (PRF) and stop codon readthrough are two translational recoding mechanisms utilized by some RNA viruses to express their structural and enzymatic proteins at a defined ratio. Efficient recoding usually requires an RNA pseudoknot located several nucleotides downstream from the recoding site. To assess the strategic importance of the recoding pseudoknots, we have carried out a large scale genome-wide analysis in which we used an in-house developed program to detect all possible H-type pseudoknots within the genomic mRNAs of 81 animal viruses. Pseudoknots are detected downstream from ~85% of the recoding sites, including many previously unknown pseudoknots. ~78% of the recoding pseudoknots are the most stable pseudoknot within the viral genomes. However, they are not as strong as some designed pseudoknots that exhibit roadblocking effect on the translating ribosome. Strong roadblocking pseudoknots are not detected within the viral genomes. These results indicate that the decoding pseudoknots have evolved to possess optimal stability for efficient recoding. We also found that the sequence at thegag-polframeshift junction of HIV1 harbors potential elaborated pseudoknots encompassing the frameshift site. A novel mechanism is proposed for possible involvement of the elaborated pseudoknots in the HIV1 PRF event.

Improving curriculum structure is critical for enhancing student success and on-time graduation, yet few methods exist to evaluate how prerequisite paths shape student progression and graduation outcomes. This study proposes a data-driven, graph-based framework that integrates course prerequisite networks with student performance data to systematically analyze curricular structure and student outcomes. We identify high-risk courses by jointly modeling their structural importance and pass rates, and quantify the time and survivability of different prerequisite paths using probabilistic models. Additionally, we introduced grade transition patterns to capture more nuanced transitions in student performance and pinpoint bottlenecks along prerequisite paths. Applying the model on four science and engineering majors from a public institution, the results not only identify high-risk courses often missed in conventional analyses, but also reveal path-level disparities and structural bottlenecks that affect student progression and time to graduation. For example, in the Computer Science major, we identified that the architecture and operating systems pathway is more challenging than the software engineering pathway. A closer examination of the course pairs along this trajectory revealed that the difficulty stems from a significant drop in student performance between a prerequisite–successor course pairs.This type of analysis fills a gap in conventional curriculum studies, which often overlook path-level dynamics, and offers actionable insights for educators a to identify high risk curricular components.

RNA pseudoknots play a crucial role in various cellular functions. Established pseudoknots show significant variation in both size and structural complexity. Specifically, three-stemmed pseudoknots are characterized by an additional stem-loop embedded in their structure. Recent findings highlight these pseudoknots as bacterial riboswitches and potent stimulators for programmed ribosomal frameshifting in RNA viruses like SARS-CoV2. To investigate the possible presence of functional three-stemmed pseudoknots in human mRNAs, we employed in-house developed computational methods to detect such structures within a dataset comprising 21,780 full-length human mRNA sequences. Numerous three-stemmed pseudoknots were identified. A selected set of 14 potential instances are presented, in which the start codon of the mRNA is found in close proximity either upstream, downstream, or within the identified three-stemmed pseudoknot. These pseudoknots likely play a role in translational initiation regulation. The probability of their existence gains support from their ranking as the most stable pseudoknot identified in the entire mRNA sequence, structural conservation across homologous mRNAs, stereochemical feasibility as demonstrated by structural modeling, and classification as members of the CPK-1 pseudoknot family, which includes many well-established pseudoknots. Furthermore, in four of the mRNAs, two or three closely spaced or tandem three-stemmed pseudoknots were identified. These findings suggest the frequent occurrence of three-stemmed pseudoknots in human mRNAs. A stepwise co-transcriptional folding mechanism is proposed for the formation of a three-stemmed pseudoknot structure. Our results not only provide fresh insights into the structures and functions of pseudoknots but also unveil the potential to target pseudoknots for treating human diseases.

Pseudoknots assume various functions including stimulation of -1 programmed ribosomal frameshifting (PRF) or stop codon readthrough (SCR) in RNA viruses. These pseudoknots vary greatly in sizes and structural complexities. Recent biochemical and structural studies confirm the three-stemmed pseudoknots as the -1 PRF stimulators in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and related coronaviruses. We reexamined previously reported -1 PRF or SCR stimulating pseudoknots, especially those containing a relatively long connecting loop between the two pseudoknot-forming stems, for their ability to form elaborated structures. Many potential elaborated pseudoknots were identified that contain one or more of the following extra structural elements: stem-loop, embedded pseudoknot, kissing hairpins, and additional loop-loop interactions. The elaborated pseudoknots are found in several different virus families that utilize either the -1 PRF or SCR recoding mechanisms. Model-building studies were performed to not only establish the structural feasibility of the elaborated pseudoknots but also reveal potential additional structural features that cannot be readily inferred from the predicted secondary structures. Some of the structures, such as embedded double pseudoknots and compact loop-loop pseudoknots mediated by the previously established common pseudoknot motif-1 (CPK-1), represent the first of its kind in the literatures. By advancing discovery of new functional RNA structures, we significantly expand the repertoire of known elaborated pseudoknots that could potentially play a role in -1 PRF and SCR regulation. These results contribute to a better understanding of RNA structures in general, facilitating the design of engineering RNA molecules with certain desired functions.Communicated by Ramaswamy H. Sarma.

It is well-established that viral and cellular mRNAs alike harbour functional long-range intra-molecular RNA-RNA interactions. Despite the biological importance of such interactions, their identification and characterization remain challenging. Here we present a computational method for the identification of certain kinds of long-range intra-molecular RNA-RNA interactions involving the loop nucleotides of a hairpin loop. Using the computational method, we analysed 4272 HIV-1 genomic mRNAs. A potential long-range intra-molecular RNA-RNA interaction within the HIV-1 genomic RNA was identified. The long-range interaction is mediated by a kissing loop structure between two stem-loops of the previously reported SHAPE-based secondary structure of the entire HIV-1 genome. Structural modelling studies were carried out to show that the kissing loop structure not only is sterically feasible, but also contains a conserved RNA structural motif often found in compact RNA pseudoknots. The computational method should be generally applicable to the identification of potential long-range intra-molecular RNA-RNA interactions in any viral or cellular mRNA sequence. Communicated by Ramaswamy H. Sarma